Many crucial biological functions are mediated or accomplished by biomolecules and tissue structures that are intrinsically fluorescent. As a result, there is an opportunity to diagnose and study important biological events by measuring and localizing the spectra and tissue fluorescence emission. To investigate in vivo internal processes and structures in large organisms, such as human beings and agricultural animals, endoscopic procedures which penetrates body cavities or even solid tissue may be required.
Endoscopy video imaging in body cavities ordinarily utilizes back-scattered white light applied through the endoscope to form a low-resolution color image of the internal surfaces of these cavities. Physicians often use the changes in shapes and changes in local apparent color (which are often due to changes in blood distribution) to recognize disease states, such as malignant tumors or inflammation. Unfortunately, these clues are frequently not sufficient, especially for detection of the early onset of disease. Diagnostic improvements have been made by quantitative measurements of the light scattering and of tissue fluorescence emission.
Ordinarily, the light required to excite the fluorescence of tissue is delivered through an optical fiber or fiber bundle that is inserted through a small tube built into the endoscopic pipe to accommodate a mechanical biopsy wire. Small optical fibers or fiber bundles can be passed easily through the same tube. Some of the strongest tissue fluorescence usually seen in this procedure are due to NADH (nicotinamide adenine dinucleotide) and to collagen structures. Their fluorescence is excited by absorption of ultraviolet light of about 300 to 400 nm wavelength corresponding to photon energies of around 3 to 4 eV or sometimes slightly longer wavelength visible light.
A first problem is that this ultraviolet light is strongly absorbed by hemoglobin and oxyhemoglobin in the blood, which are not fluorescent, so that penetration of the illumination into the tissue depends on their concentration and distribution.
A second problem is that the illumination exiting the optical fibers into tissue fans out at an included angle determined by the numerical aperture (NA) of the optical fiber. Small lenses can focus the spread so that the light first converges to a focus but it then fans out beyond the focal plane. (Typically, the NA is about 0.2 and the included cone angle is ˜17°.) This angular spreading is a problem, because roughly equal total amounts of fluorescence are excited in every spherical section at each distance from the end of the fiber until attenuated by absorption. This effect is schematically illustrated in FIG. 1. Fluorescence excitation is similarly spread out. Scattering does not attenuate the fluorescence excitation but does distribute it even more broadly. Consequently, the volume observed is ill defined with its practical limits depending also on blood distribution and light scattering. It should be noted that these problems tend to persist even if lenses focus the illumination and/or prisms and mirrors deflect the light for side viewing.
The present invention is directed to overcoming these deficiencies in the art.